Hidetaka Takeda

Role of nitric oxide in regulation of cerebral microvascular tone and autoregulation of cerebral blood flow in cats.

The role of nitric oxide in the regulation of cerebrocortical microvascular tone and autoregulation of cerebral blood flow (CBF) was examined in 24 anesthetized cats. The local cerebral blood volume (CBV), mean transit time of blood (MTT), and CBF in the cortex were measured by our photoelectric method. CBV represents the cumulative dimensions of the cerebral microvessels. Intravenous injection of 0.35-0.7 mg/kg/min NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthesis, significantly increased mean arterial blood pressure (MABP; 8.4-14.1%, P < 0.01), decreased CBV (15.2-28.7%, P < 0.01), and decreased CBF (20.0-29.8%, P < 0.01) in a dose-related manner. The changes in MABP, CBV, and CBF elicited by L-NMMA were inhibited (P < 0.05) by simultaneous infusion of 35 mg/kg/min L-arginine. Autoregulation of CBF was examined during controlled hypotension of -30 to -40 mmHg (artificial bleeding) and recovery of blood pressure (reinfusion of blood). Although CBF remained constant with blood pressure changes in the control state (delta CBF/delta MABP of 0.037 +/- 0.155 with hypotension), CBF became dependent on blood pressure changes (delta CBF/delta MABP of 0.478 +/- 0.135, P < 0.05) during infusion of 0.35 mg/kg/min L-NMMA. It is concluded that nitric oxide participates in both the regulation of basal tone of cerebral microvessels and the autoregulation of CBF.

Initial oligemia with capillary flow stop followed by hyperemia during K+-induced cortical spreading depression in rats

Local cerebral blood volume (CBV) and capillary flow changes in regions of depolarizing neurons during K+-induced cortical spreading depression (CSD) in the cerebral cortex of α-chloralose-urethane-anesthetized rats were examined employing a transillumination (550 nm) video system. Capillary flow was calculated as the reciprocal of mean transit times of blood in pixels of 40 μm × 40 μm, each of which contains a few capillaries. Potassium microinjection into the cortex evoked repetitive wave-ring spreads of oligemia at a speed of ca. 2.33±0.48 mm/min. During the spread of CSD, tracer (either saline or carbon black) was injected into the internal carotid artery. Colocated with the oligemic wave, we detected capillary flow stop as evidenced by disappearance of the hemodilution curves. At any location in the region of interest within the cerebral cortex, we observed cyclic changes of capillary flow stop/hyperperfusion in synchrony with oligemia/hyperemia fluctuations. The initial flow stop and oligemia were ascribed to capillary compression by astroglial cell swelling, presumably at the pericapillary endfeet, since the oligemia occurred before larger vessel changes. We conclude that local depolarizing neurons can decrease adjacent capillary flow directly and immediately, most likely via astroglial cell swelling, and that the flow stop triggers upstream arteriolar dilatation for capillary hyperperfusion.

Transient cerebral vasodilatory effect of neuropeptide y mediated by nitric oxide

The effects of intracarotidly injected neuropeptide Y (NPY; 0.1 micrograms/kg) on the local cerebral blood volume (CBV) and blood flow (CBF) in the parieto-temporal cortex were examined by the photoelectric method in 17 anesthetized cats. CBV reflects the cumulative crosssectional area of the cerebral microvascular beds. NPY immediately caused transient but significant increases in CBV and CBF, which lasted for less than 5 min. Thereafter, CBV returned to and remained at the control level, although CBF was decreased by 30-40% for 60 min during the monitoring period. The CBV increases after NPY were prevented by a 15-min preinjection of 0.35 mg/kg/min of NG-monomethyl-L-arginine (L-NMMA), which is a competitive blocker of nitric oxide synthesis. The CBV increases after NPY reappeared following a 15-min administration of 0.25 mg/kg/min of L-arginine, which is a precursor of nitric oxide. We conclude that NPY administered in vivo exerts a previously unreported effect of transient vasodilatation on the cerebral microvessels. This action appears to be mediated by nitric oxide, which is a major candidate as an endothelium-derived relaxing factor (EDRF).

Repetitive concentric wave-ring spread of oligemia/hyperemia in the sensorimotor cortex accompanying K+-induced spreading depression in rats and cats

Vascular changes accompanying spreading depression (SD) remain controversial. We examined dynamic alterations of local cerebral blood volume (CBV) during SD by observing light transmission at an isosbestic point of hemoglobin (550 nm) in seven rats and five cats under alpha-chloralose/urethane anesthesia. The two species were used for comparison between the lissencephalic and gyrencephalic brains. We found that a concentrated K(+) solution microinjected into the sensorimotor cortex provoked CBV changes that appeared as a repetitive propagation of concentric wave-rings of ischemia followed by hyperemia expanding peripherally from the injection site at speeds of 1.9-3.2 mm/min. The dynamic CBV changes continued repeatedly every 1-5 min for more than 30 min in three rats, ceased within 30 min in three rats and remained at the site of K(+) injection in one rat. Similar repeated CBV changes occurred in two out of five cats.

Dilatation of cerebral microvessels mediated by endothelin ETB receptor and nitric oxide in cats

The functional role of the endothelin (ET)B receptor in the cerebral microvessels was examined in seven anesthetized cats. The effects of intracarotidly injected IRL1620, a selective ETB receptor agonist, on the cerebral blood volume (CBV) in the cortex were examined by a photoelectric method. CBV reflects the cumulative dimensions of the cerebral microvessels. High doses of IRL1620 (0.1-1 nmol/kg) elicited a transient fall in CBV, followed by a marked and dose-related increase of CBV (P < 0.01). The CBV increase was significantly inhibited (P < 0.01) by simultaneous administration of 0.35 mg/kg/min NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthesis. The marked, but transient, depressor effect of IRL1620 was also attenuated (P < 0.01) by L-NMMA. The ETB receptor subtype and production of nitric oxide, probably in the endothelium, mediate the vasodilatory action of ET in the cerebral microvessels.

Hydrogen peroxide enhances phagocytic activity of ameboid microglia

Microglia are considered to serve as a guardian of the brain. In achieving this task, they have been observed to transform into a reactive form and then an ameboid form. Several substances are implicated in the control of such behavior. We examined the effect of hydrogen peroxide on cultured microglia of ameboid form obtained from the fetal rat brain employing video-enhanced contrast-differential interference contrast microscopy. Microglia harvested from the culture bottle were observed to float in a spherical shape with abundant filopodia on the surface. However, on coming into contact with the glass surface of a cover slip, they immediately transformed into an ameboid form. The microglia spread themselves out, surrounded by thin transparent lammellipodia, which would not be clearly observable by either light microscopy or electron microscopy. In a concentration-dependent fashion, 10(-3)-10(-1) M hydrogen peroxide solution enhanced the ruffling process of the lamellipodia and formation of vesicles (phagosomes), which displayed a typical phagocytotic form. It is concluded that an increase in free radicals in ischemic tissue tends to facilitate the phagocytosis of ameboid microglia as macrophages.

Constriction/Dilatation of the Cerebral Microvessels by Intravascular Endothelin-1 in Cats

The effects of intracarotidly injected endothelin (ET)-1 (0.01-3 nmol) on the local cerebral blood volume (CBV) in the parietotemporal cortex were examined by the photoelectric method in 17 anesthetized cats. CBV reflects the cumulative dimensions of the cerebral microvessels. Low doses of ET-1 (0.01 and 0.1 nmol) elicited mild but significant reductions in CBV without changes in the systemic arterial blood pressure (SABP). High doses of ET-1 (3 nmol) initially induced marked declines of CBV, which were attributable to the significant falls in SABP. CBV subsequently exhibited significant increases. The CBV increases were not secondary to the accompanying elevations of SABP, since they were unaffected by inhibition of the SABP changes after preinjection of BQ-123 (1 mg/kg), an ET antagonist specific to the ETA receptors. The CBV increases, however, were prevented by continuous administration of NG-monomethyl-L-arginine (0.35 mg/kg/min), an inhibitor of nitric oxide synthesis, plus BQ-123. We conclude that while low doses of intravascular ET-1 constrict the cerebral microvessels, high doses of ET-1 dilate the cerebral microvessels through the induction of nitric oxide probably in the cerebrovascular endothelium.

Non-amoeboid locomotion of cultured microglia obtained from newborn rat brain

The movements of cultured microglia obtained from newborn rat brain were examined by video enhanced-differential interference contrast (VEC-DIC) microscopy. Active microglia measured 9.03 +/- 1.06 microns in diameter (mean +/- SD, n = 33; range, 7.03-10.36 microns). The microglia appeared to become smaller with spread of lamellipodia. The short axis of actively moving microglia measured 7.03 +/- 0.49 microns (n = 7). The lamellipodia were thin, transparent and developed rapidly around the cell body (maximal speed of extension, 4 or 5 microns/s). When shear stress from the medium was applied to the surface of cultured cells, the microglia swam with flat lamellipodia serving as sails in the stream. Spontaneous non-amoeboid movements of microglia were observed: they pivoted, circled and marched in various directions using their lamellipodia. The angular speed of rotation was maximally 3 degrees/s. In 5 marching cells, the average speed (distance/s) was calculated at 1.01 +/- 0.54 microns/s (ca. 60 microns/min or 3.6 microns/h).

Effects of sumatriptan on the cerebral intraparenchymal microcirculation in the cat.

1 Sumatriptan, a 5‐hydroxytryptamine (5‐HT)1‐like receptor agonist, is effective against the headache of migraine. The effects of sumatriptan injected via the carotid artery on the cerebral microcirculation were studied in 10 anaesthetized cats. 2 The local cerebral blood volume (CBV), mean transit time of blood (MTT) and cerebral blood flow (CBF) in the parieto‐temporal cortex were measured by a photoelectric method. CBV represents the cumulative dimensions of the cerebral microvessels. 3 Sumatriptan at 5 and 50 μg kg−1 had no significant effects on the CBV, MTT, CBF, and mean arterial blood pressure (MABP); 500 μg kg−1 of sumatriptan reduced the CBV, prolonged the MTT, and decreased the CBF (approximately −20%) without affecting the MABP. Sumatriptan, 5 mg kg−1, elicited transient reductions in CBV and CBF, which were attributable to the rapid and marked falls of MABP seen with this dose. 4 Thus, while a high dose of sumatriptan (500 μg kg−1) exhibits direct vasoconstrictor actions on the cerebral vessels, low doses of sumatriptan, within the therapeutic range, elicit no vasoconstriction. The data do not support a vasoconstrictor action of sumatriptan playing a primary role in reversing the headache of migraine.

Platelet adhesion to aortic endothelial cells in vitro after thrombin treatment : observation with video-enhanced contrast microscopy

Secondary thrombus formation following arterial occlusion is suggested to play an important role in the exacerbation of ischemic organ damage. We investigated the effect of thrombin on endothelial cells from the aspect of morphological changes and induction of platelet adhesion to the endothelial cells. Using a video-enhanced contrast microscopy, we observed human aortic endothelial cells (HAEC) following perfusion of human alpha-thrombin of 1.0 U/ml (n = 7) or vehicle (n = 7) for 30 minutes. The endothelial cells began to shrink 15 minutes after thrombin administration. Gaps between the cells were formed. The cells became rearranged orderly in the same direction 30 minutes later. In another study, following pretreatment with human alpha-thrombin 1.0 U/ml (n = 10) or vehicle (n = 7) for 20 minutes and washout, platelets were perfused over HAEC for 30 minutes. Platelets adhered directly to thrombin-treated endothelial cells and became flat on the endothelial cells. Then other platelets were observed to approach to the flattened platelets and aggregated onto it. After washout of floating platelets, adhesion of platelets was further confirmed. These results suggest that thrombin may be involved in the endothelial damage and formation of platelet thrombi on the endothelial cells after blood flow disturbance.